The phosphorylated form of rat pp63, a hepatic glycoprotein, selectively inhibits both insulin receptor tyrosine kinase activity (IR-TKA) and insulin-mediated growth promotion, but does not affect several other insulin-mediated activities. Our new data suggest that the human protein, alpha2-HS-glycoprotein (alpha2-HSG), in its phosphorylated form, functions similarly to rat pp63 in inhibiting IR autophosphorylation, IR-TKA and insulin-dependent mitogenesis in a dose-dependent fashion, non-competitive with respect to insulin. Since our long-term objective is to investigate the role of IR-TKA in both the normal and insulin-resistant states of rats and humans, this dissection of insulin-mediated cellular functions, by pp63/alpha2-HSG, provides us with a novel approach. Using this natural IR-TKA inhibitor, we can now examine the role of IR-TKA in the delivery of specific insulin-mediated signals. Our specific aims include: (1) Investigation of effects of pp63/alpha2-HSG on metabolic and mitogenic actions of insulin and on functions of the insulin receptor in a rat hepatoma cell line, Epstein-Barr Virus transformed lymphocytes and transfected rat fibroblasts expressing normal insulin receptors, (2) Examination of synthesis and secretion of pp63/alpha2-HSG and characterization of specific activity of circulating pp63/alpha2-HSG in rat and human insulin resistant states in vivo (high-fat diet and obesity, respectively), (3) Examination of mechanisms underlying these observations by utilizing human insulin receptor mutants as well as monoclonal antibodies to the insulin receptor and to pp63/alpha2-HSG for modifying pp63/alpha2-HSG - insulin receptor interaction. Our preliminary data demonstrate that pp63 gene expression directly correlates with the degree of insulin resistance, in a rat high-fat diet model. These date support our contention that studies on regulation of pp63/alpha2-HSG specific activity will yield information critical to our understanding of impairment of insulin signaling in insulin-resistant conditions (diabetes mellitus, hypertension, obesity, and dyslipidemia).